The ubiquitination of many cell surface regulatory proteins is critical for their downregulation because ubiquitin signals control internalization and sorting of proteins in the endocytic pathway. Ubiquitin protein ligases are key components of the ubiquitination machinery that recognize and modify specific plasma membrane substrates at the appropriate time to initiate downregulation. These ligases play multiple roles in endocytosis: they are required to ubiquitinate endocytic cargo, they modify trans-acting components of the endocytic machinery, and they control transport of proteins later in the endocytic pathway. When plasma membrane proteins cannot be recognized and modified by ubiquitin protein ligases, human diseases, such as cancers and an inherited form of hypertension (Liddls syndrome), occur. The Nedd4/Rsp5p family of hect ubiquitin protein ligases modify and downregulate signaling receptors, channels and permeases. Nedd4/Rsp5p carries a C2 domain, which may be involved in Ca+2-dependent interactions with phospholipids or proteins, three WW protein-protein interaction motifs, and a catalytic hect domain. Nedd4 is required for ubiquitination and endocytosis of the epithelial sodium channel. Other C2-WW-hect domain ligases ubiquitinate and/or regulate Notch and TGFB receptors. The yeast homologue of these ligases, Rsp5p, ubiquitinates phosphorylated plasma membrane proteins to trigger rapid endocytosis. We have recently shown that trans-acting endocytic proteins that are required for internalization and endosomal transport are also ubiquitinated by Rsp5p. For example, we have identified one component of the endocytic machinery, an amphiphysin homologue called Rvsl67p, as a target of Rsp5p-dependent monoubiquitination. The experiments described in this grant are proposed to define the roles of hect domain ubiquitin protein ligases at multiple steps in the endocytic pathway that lead to downregulation and degradation of cell surface regulatory proteins. The aims of the grant are 1) to define how the Rsp5p ubiquitin protein ligase recognizes and ubiquitinates phosphorylated receptors; 2) to identify trans-acting factors of the endocytic machinery that are regulated by Rsp5p-dependent ubiquitination; and 3) to define the cellular and molecular functions of Rsp5p-dependent monoubiquitination of the amphiphysin homologue, Rvsl67p.